Synthesis and characterization of recombinant, authentic human prolactin secreted into the periplasmic space of Escherichia coli.
Recombinant, fully bioactive, authentic human prolactin (aut-hPRL) has been synthesized in transformed Escherichia coli HB2151 bacteria in a soluble, non-glycosylated form, which is secreted into the bacterial periplasm. Use was made of a bacterial expression vector, containing tac promoter-controlled sequences for the translation enhancer from bacteriophage T7 gene 10, and for a cellulase leader peptide from Cellulomonas fimi joined to sequences coding for aut-hPRL. This vector was derived from a previously described vector containing sequences of an hPRL variant, tag-hPRL (containing a 12-amino-acid peptide tag at the N-terminal end), using site-specific mutagenesis to delete the tag sequence. SDS/PAGE, partial N-terminal amino acid sequence analysis, Western blot analysis and Nb2 lymphoma cell in vitro bioassay indicated correct processing of the hormone. Periplasmic secretion of aut-hPRL, as measured by immunoassay, was relatively low (approx. 0.08 microgram/ml per A600 unit), in contrast to that of tag-hPRL which was approximately 8-fold higher, apparently a consequence of the tag sequence. This is the first report describing periplasmic secretion of biologically active, authentic hPRL.
Morganti
L
L
Biotechnology Department, National Nuclear Energy Commission (IPEN-CNEN), Cidade Universitária, São Paulo, Brazil.
Soares
C R
CR
Affonso
R
R
Gout
P W
PW
Bartolini
P
P
D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000235:genetics; D015153:Blotting, Western; D002480:Cellulase / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D008223:Lymphoma; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019897:Periplasm / Q000378:metabolism*; D011388:Prolactin / Q000096:biosynthesis* / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000737:chemistry; D014407:Tumor Cells, Cultured
D011994:Recombinant Proteins; D011388:Prolactin; D002480:Cellulase